Histopathological Observation of Immunized Rhesus Macaques with Plague Vaccines after Subcutaneous Infection of Yersinia pestis

In our previous study, complete protection was observed in Chinese-origin rhesus macaques immunized with SV1 (20 µg F1 and 10 µg rV270) and SV2 (200 µg F1 and 100 µg rV270) subunit vaccines and with EV76 live attenuated vaccine against subcutaneous challenge with 6×106 CFU of Y. pestis. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological and immunohistochemical techniques. In addition, the glomerular basement membranes (GBMs) of the immunized animals and control animals were checked by electron microscopy. The results show no signs of histopathological lesions in the lungs, livers, kidneys, lymph nodes, spleens and hearts of the immunized animals at Day 14 after the challenge, whereas pathological alterations were seen in the corresponding tissues of the control animals. Giemsa staining, ultrastructural examination, and immunohistochemical staining revealed bacteria in some of the organs of the control animals, whereas no bacterium was observed among the immunized animals. Ultrastructural observation revealed that no glomerular immune deposits on the GBM. These observations suggest that the vaccines can effectively protect animals from any pathologic changes and eliminate Y. pestis from the immunized animals. The control animals died from multi-organ lesions specifically caused by the Y. pestis infection. We also found that subcutaneous infection of animals with Y. pestis results in bubonic plague, followed by pneumonic and septicemic plagues. The histopathologic features of plague in rhesus macaques closely resemble those of rodent and human plagues. Thus, Chinese-origin rhesus macaques serve as useful models in studying Y. pestis pathogenesis, host response and the efficacy of new medical countermeasures against plague

Cloning and Expression of Multiple Cytochrome P450 Genes: Induction by Fipronil in Workers of the Red Imported Fire Ant (Solenopsis invicta Buren).

Both exogenous and endogenous compounds can induce the expression of cytochrome P450 genes. The insect cytochrome P450 genes related to insecticide resistance are likely to be expressed as the "first line of defense" when challenged with insecticides. In this study, four cytochrome P450 genes, SinvCYP6B1, SinvCYP6A1, SinvCYP4C1, and SinvCYP4G15, were firstly isolated from workers of the red imported fire ant (Solenopsis invicta) through rapid amplification of cDNA ends (RACE) and sequenced. The fipronil induction profiles of the four cytochrome P450 genes and the two previously isolated CYP4AB1 and CYP4AB2 were characterized in workers. The results revealed that the expression of SinvCYP6B1, SinvCYP6A1, CYP4AB2, and SinvCYP4G15, increased 1.4-fold and 1.3-fold more than those of acetone control, respectively, after 24 h exposure to fipronil at concentrations of 0.25 μg mL-1 (median lethal dose) and 0.56 μg mL-1 (90% lethal dose), while no significant induction of the expression of CYP4AB1 and SinvCYP4C1 was detected. Among these genes, SinvCYP6B1 was the most significantly induced, and its maximum expression was 3.6-fold higher than that in acetone control. These results might suggest that multiple cytochrome P450 genes are co-up-regulated in workers of the fire ant through induction mechanism when challenged with fipronil. These findings indicated that cytochrome P450 genes play an important role in the detoxification of insecticides and provide a theoretical basis for the mechanisms of insecticide metabolism in the fire ant

Relative expression levels of <i>SinvCYP6B1</i>, <i>SinvCYP6A1</i>, <i>SinvCYP4C1</i>, <i>SinvCYP4G15</i>, <i>CYP4AB1</i>, and <i>CYP4AB2</i> in workers of the fire ant following treatment with fipronil at the LC₅₀ (0.25 μg mL⁻¹).

<p>Relative expression levels of <i>SinvCYP6B1</i>, <i>SinvCYP6A1</i>, <i>SinvCYP4C1</i>, <i>SinvCYP4G15</i>, <i>CYP4AB1</i>, and <i>CYP4AB2</i> in workers following treatment with 0.25 μg mL⁻¹ fipronil at 12, 24, 36, and 48 h (60 and 72 h) were determined by qRT-PCR. The experiments were repeated three times. The results are presented as the mean ± S.E. Significant differences within time points are indicated by *(P<0.05).</p

Time-dependent mortality of workers exposed to fipronil at 0.25 μg mL⁻¹ and 0.56 μg mL⁻¹ after 12, 24, 36, 48, 60, and 72 h.

<p>Data are presented as the mean ± standard error (S.E.) of three independent replicates. Different letters after the standard errors indicate significant differences among treatments based on ANOVA followed by Tukey’s multiple comparison test (p<0.05) for the same time point.</p

Phylogenetic relationships among the CYP4s and CYP6s from the fire ant.

<p>The CYPs are presented with their GenBank accession numbers. The un-rooted phylogenetic tree was constructed using the neighbor-joining method. Nodes indicate bootstrap values calculated with 1000 replicates.</p

Alignment of the deduced amino acid sequences of SinvCYP6B1, SinvCYP6A1, SinvCYP4C1, and SinvCYP4G15 with CYP4AB1 and CYP4AB2 from the fire ant.

<p>Amino acid residues that were conserved among all four sequences and residues that were present in more than two P450 proteins are indicated by blue boxes. Invariant and highly conserved motifs in the P450 amino acid sequences are highlighted in red boxes.</p

Long-Term Observation of Subunit Vaccine F1-rV270 against Yersinia pestis in Mice▿ †

Long-term protection and antibody response for the subunit vaccine F1-rV270 were determined by using the mouse model. Antibodies to F1 and rV270 were still detectable over a period of 518 days. The complete protection against lethal challenge of Yersinia pestis could be achieved up to day 518 after primary immunization

The new economics of the business case for sustainability

Source tracing of pathogens is critical for the control and prevention of infectious diseases. Genome sequencing by high throughput technologies is currently feasible and popular, leading to the burst of deciphered bacterial genome sequences. Utilizing the flooding genomic data for source tracing of pathogens in outbreaks is promising, and challenging as well. Here, we employed Yersinia pestis genomes from a plague outbreak at Xinghai county of China in 2009 as an example, to develop a simple two-step strategy for rapid source tracing of the outbreak. The first step was to define the phylogenetic position of the outbreak strains in a whole species tree, and the next step was to provide a detailed relationship across the outbreak strains and their suspected relatives. Through this strategy, we observed that the Xinghai plague outbreak was caused by Y. pestis that circulated in the local plague focus, where the majority of historical plague epidemics in the Qinghai-Tibet Plateau may originate from. The analytical strategy developed here will be of great help in fighting against the outbreaks of emerging infectious diseases, by pinpointing the source of pathogens rapidly with genomic epidemiological data and microbial forensics information